scholarly journals Transcription-mediated organization of the replication initiation program across large genes sets up common fragile sites genome-wide

2019 ◽  
Author(s):  
Olivier Brison ◽  
Sami EL-Hilali ◽  
Dana Azar ◽  
Stéphane Koundrioukoff ◽  
Mélanie Schmidt ◽  
...  
Keyword(s):  
Replication Timing ◽  
S Phase ◽  
Fragile Sites ◽  
Replication Initiation ◽  
Common Fragile Sites ◽  
Large Gene ◽  
Genome Wide ◽  
Underlying Mechanisms ◽  
Large Genes ◽  
Nascent Transcripts

ABSTRACTCommon Fragile Sites (CFSs) are chromosome regions prone to breakage under replication stress, known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription elicits their instability but the underlying mechanisms remained elusive. Analyses of genome-wide replication timing of human lymphoblasts here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, thus replicated by long-traveling forks. In contrast to formation of sequence-dependent fork barriers or head-on transcription-replication conflicts, traveling-long in late S phase explains CFS replication features. We further show that transcription inhibition during the S phase, which excludes the setting of new replication origins, fails to rescue CFS stability. Altogether, results show that transcription-dependent suppression of initiation events delays replication of large gene body, committing them to instability.

scholarly journals Transcription-mediated organization of the replication initiation program across large genes sets common fragile sites genome-wide

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Olivier Brison ◽  
Sami El-Hilali ◽  
Dana Azar ◽  
Stéphane Koundrioukoff ◽  
Mélanie Schmidt ◽  
...  
Keyword(s):  
Replication Timing ◽  
S Phase ◽  
Fragile Sites ◽  
Replication Initiation ◽  
Common Fragile Sites ◽  
Large Gene ◽  
Genome Wide ◽  
Underlying Mechanisms ◽  
Large Genes ◽  
Nascent Transcripts

AbstractCommon fragile sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription–replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription–replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability.

scholarly journals Transcription-dependent regulation of replication dynamics modulates genome stability

2018 ◽  
Author(s):  
Marion Blin ◽  
Benoît Le Tallec ◽  
Viola Naehse ◽  
Mélanie Schmidt ◽  
Gael A. Millot ◽  
...  
Keyword(s):  
Genome Stability ◽  
Chromosomal Rearrangements ◽  
Replication Timing ◽  
Replication Stress ◽  
S Phase ◽  
Fragile Sites ◽  
Replication Initiation ◽  
Replication Gene ◽  
Large Genes ◽  
Dynamics Analyses

Replication stress is a primary threat to genome stability and has been implicated in tumorigenesis1, 2. Common fragile sites (CFSs) are loci hypersensitive to replication stress3 and are hotspots for chromosomal rearrangements in cancers4. CFSs replicate late in S-phase3, are cell-type dependent4–6 and nest within very large genes4, 7–9. The mechanisms responsible for CFS instability are still discussed, notably the relative impact of transcription-replication conflicts7, 8, 10versus their low density in replication initiation events5, 6. Here we address the relationships between transcription, replication, gene size and instability by manipulating the transcription of three endogenous large genes, two in chicken and one in human cells. Remarkably, moderate transcription destabilises large genes whereas high transcription levels alleviate their instability. Replication dynamics analyses showed that transcription quantitatively shapes the replication program of large genes, setting both their initiation profile and their replication timing as well as regulating internal fork velocity. Noticeably, high transcription levels advance the replication time of large genes from late to mid S-phase, which most likely gives cells more time to complete replication before mitotic entry. Transcription can therefore contribute to maintaining the integrity of some difficult-to-replicate loci, challenging the dominant view that it is exclusively a threat to genome stability.

scholarly journals Global landscape of replicative DNA polymerase usage in the human genome

2021 ◽  
Author(s):  
Eri Koyanagi ◽  
Yoko Kakimoto ◽  
Fumiya Yoshifuji ◽  
Toyoaki Natsume ◽  
Atsushi Higashitani ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Division Of Labour ◽  
Fragile Sites ◽  
Replication Initiation ◽  
Genome Wide ◽  
Leading Strand ◽  
Replicative Polymerases ◽  
Large Genes ◽  
Lagging Strand ◽  
Hct116 Cells

The division of labour between DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequence (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combing the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality, zones of replication initiation and termination. We confirm that transcriptional activity shapes the patterns of initiation and termination and, by separately analysing the effect of transcription on both co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands in both co-directional and convergent forks is compromised by transcription. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability.

scholarly journals High-throughput analysis of DNA replication in single human cells reveals constrained variability in the location and timing of replication initiation

2021 ◽  
Author(s):  
Dashiell J Massey ◽  
Amnon Koren
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Replication Timing ◽  
S Phase ◽  
Human Cells ◽  
Replication Initiation ◽  
High Throughput Analysis ◽  
Timing Variability ◽  
Fixed Set ◽  
Fixed Order

DNA replication occurs throughout the S phase of the cell cycle, initiating from replication origin loci that fire at different times. Debate remains about whether origins are a fixed set of loci used across all cells or a loose agglomeration of potential origins used stochastically in individual cells, and about how consistent their firing time during S phase is across cells. Here, we develop an approach for profiling DNA replication in single human cells and apply it to 2,305 replicating cells spanning the entire S phase. The resolution and scale of the data enabled us to specifically analyze initiation sites and show that these sites have confined locations that are consistently used among individual cells. Further, we find that initiation sites are activated in a similar, albeit not fixed, order across cells. Taken together, our results suggest that replication timing variability is constrained both spatially and temporally, and that the degree of variation is consistent across human cell lines.

scholarly journals Oncogene-induced replication stress preferentially targets common fragile sites in preneoplastic lesions. A genome-wide study

Oncogene ◽  
2007 ◽  
Vol 27 (23) ◽  
pp. 3256-3264 ◽  
Author(s):  
P K Tsantoulis ◽  
A Kotsinas ◽  
P P Sfikakis ◽  
K Evangelou ◽  
M Sideridou ◽  
...  
Keyword(s):  
Replication Stress ◽  
Fragile Sites ◽  
Preneoplastic Lesions ◽  
Common Fragile Sites ◽  
Genome Wide ◽  
A Genome ◽  
Genome Wide Study

scholarly journals Xenopus Cdc7 executes its essential function early in S phase and is counteracted by checkpoint-regulated protein phosphatase 1

Open Biology ◽  
2014 ◽  
Vol 4 (1) ◽  
pp. 130138 ◽  
Author(s):  
Wei Theng Poh ◽  
Gaganmeet Singh Chadha ◽  
Peter J. Gillespie ◽  
Philipp Kaldis ◽  
J. Julian Blow
Keyword(s):  
Protein Phosphatase ◽  
Replication Timing ◽  
S Phase ◽  
Replication Initiation ◽  
Protein Phosphatase 1 ◽  
Checkpoint Kinases ◽  
Anti Cancer ◽  
Egg Extracts ◽  
Mcm Proteins

The initiation of DNA replication requires two protein kinases: cyclin-dependent kinase (Cdk) and Cdc7. Although S phase Cdk activity has been intensively studied, relatively little is known about how Cdc7 regulates progression through S phase. We have used a Cdc7 inhibitor, PHA-767491, to dissect the role of Cdc7 in Xenopus egg extracts. We show that hyperphosphorylation of mini-chromosome maintenance (MCM) proteins by Cdc7 is required for the initiation, but not for the elongation, of replication forks. Unlike Cdks, we demonstrate that Cdc7 executes its essential functions by phosphorylating MCM proteins at virtually all replication origins early in S phase and is not limiting for progression through the Xenopus replication timing programme. We demonstrate that protein phosphatase 1 (PP1) is recruited to chromatin and rapidly reverses Cdc7-mediated MCM hyperphosphorylation. Checkpoint kinases induced by DNA damage or replication inhibition promote the association of PP1 with chromatin and increase the rate of MCM dephosphorylation, thereby counteracting the previously completed Cdc7 functions and inhibiting replication initiation. This novel mechanism for regulating Cdc7 function provides an explanation for previous contradictory results concerning the control of Cdc7 by checkpoint kinases and has implications for the use of Cdc7 inhibitors as anti-cancer agents.

scholarly journals G2 phase chromatin lacks determinants of replication timing

2010 ◽  
Vol 189 (6) ◽  
pp. 967-980 ◽  
Author(s):  
Junjie Lu ◽  
Feng Li ◽  
Christopher S. Murphy ◽  
Michael W. Davidson ◽  
David M. Gilbert
Keyword(s):  
Spatial Organization ◽  
Replication Timing ◽  
S Phase ◽  
Decision Point ◽  
Genome Wide Analysis ◽  
Quiescent Cells ◽  
Egg Extract ◽  
Genome Wide ◽  
G2 Phase

DNA replication in all eukaryotes follows a defined replication timing program, the molecular mechanism of which remains elusive. Using a Xenopus laevis egg extract replication system, we previously demonstrated that replication timing is established during early G1 phase of the cell cycle (timing decision point [TDP]), which is coincident with the repositioning and anchorage of chromatin in the newly formed nucleus. In this study, we use this same system to show that G2 phase chromatin lacks determinants of replication timing but maintains the overall spatial organization of chromatin domains, and we confirm this finding by genome-wide analysis of rereplication in vivo. In contrast, chromatin from quiescent cells retains replication timing but exhibits disrupted spatial organization. These data support a model in which events at the TDP, facilitated by chromatin spatial organization, establish determinants of replication timing that persist independent of spatial organization until the process of chromatin replication during S phase erases those determinants.

scholarly journals Perturbed replication induced genome wide or at common fragile sites is differently managed in the absence of WRN

2012 ◽  
Vol 33 (9) ◽  
pp. 1655-1663 ◽  
Author(s):  
I. Murfuni ◽  
A. De Santis ◽  
M. Federico ◽  
M. Bignami ◽  
P. Pichierri ◽  
...  
Keyword(s):  
Fragile Sites ◽  
Common Fragile Sites ◽  
Genome Wide

scholarly journals Replication Timing and Transcription Identifies a Novel Fragility Signature Under Replication Stress

2019 ◽  
Author(s):  
Dan Sarni ◽  
Takayo Sasaki ◽  
Karin Miron ◽  
Michal Irony Tur-Sinai ◽  
Juan Carlos Rivera-Mulia ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromosomal Instability ◽  
Temporal Order ◽  
Replication Timing ◽  
Replication Stress ◽  
Fragile Sites ◽  
Precise Mapping ◽  
Chromosomal Fragility ◽  
Transcriptional Changes ◽  
Large Genes

AbstractCommon fragile sties (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features characterizing CFSs have been associated with their instability, however, these features are prevalent across the genome and do not account for all known CFSs. Therefore, the molecular mechanism underlying CFS instability remains unclear. Here, we explored the transcriptional profile and temporal order of DNA replication (replication timing, RT) of cells under replication stress conditions. We show that the RT of only a small portion of the genome is affected by replication stress, and that CFSs are enriched for delayed RT. We identified a signature for chromosomal fragility, comprised of replication stress-induced delay in RT of early/mid S-phase replicating regions within actively transcribed large genes. This fragility signature enabled precise mapping of the core fragility region. Furthermore, the signature enabled the identification of novel fragile sites that were not detected cytogenetically, highlighting the improved sensitivity of our approach for identifying fragile sites. Altogether, this study reveals a link between altered DNA replication and transcription of large genes underlying the mechanism of CFS expression. Thus, investigating the RT and transcriptional changes in cancer may contribute to the understanding of mechanisms promoting genomic instability in cancer.

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